evaluating-leaf-responses-to-microbial-secondary-metabolites-using

Evaluating Leaf Responses to Microbial Secondary Metabolites Using A High-Throughput Format

Microbes secrete structurally diverse secondary metabolites during plant infection, some of which are detected by plant cells, which trigger stress ...

Agriculture and Agri-Food Canada

University of Ottawa

Kinases facilitate detection of extracellular signals and set in motion cellular responses for plant adaptation and survival. Some of the energy utilized for kinase signal processing is produced through the activity of ion transporters. Additionally, the synergy between cellular ions and signal transduction influences plant response to pathogens, and their growth and development. In plants, the signaling elements that connect cell wall and membrane sensors with ion homeostasis and transport-mediated processes are largely unknown. Current research indicates that plant  (s), a subfamily , may have evolved to fulfill this role. In this review, we explore new findings on plant  placing a particular focus on the connection between ILKs proteins unique structural features and ILKs functions. The ankyrin repeat motifs and the kinase domains of ILKs in  and land plants lineage, respectively, are analyzed and discussed as potential determinants of ILKs' metal ion cofactor specificity and their enzymatic and interaction activities. Further,  regulation through gene expression, subcellular localization, and ions and ion transporters is reviewed in the context of recent studies. Finally, using evidence from literature and interactomics databanks, we infer ILKs-dependent cellular pathways and highlight their potential in transmitting multiple types of signals originating at the interface between the cell wall and plasma membrane.

Insights into the Structure, Function, and Ion-Mediated Signaling Pathways Transduced by Plant Integrin-Linked Kinases.

The enzyme phytochelatin synthase (PCS) has long been studied with regard to its role in metal(loid) detoxification in several organisms, i.e., plants, yeasts, and nematodes. It is in fact widely recognized that PCS detoxifies a number of heavy metals by catalyzing the formation of thiol-rich oligomers, namely phytochelatins, from glutathione and related peptides. However, recent investigations have highlighted other possible roles played by the PCS enzyme in the plant cell, e.g., the control of pathogen-triggered callose deposition. In order to examine novel aspects of  PCS1 (AtPCS1) functions and to elucidate its possible roles in the secondary metabolism, metabolomic data of  wild-type and  mutant were compared, the latter lacking AtPCS1. HPLC-ESI-MS analysis showed differences in the relative levels of metabolites from the glucosinolate and phenylpropanoid pathways between  and wild-type plants. Specifically, in control (Cd-untreated) plants, higher levels of 4-methoxy-indol-3-ylmethylglucosinolate were found in  plants vs. wild-type. Moreover, the  mutant showed to be impaired in the deposit of callose after Cd exposure, suggesting that AtPCS1 protects the plant against the toxicity of heavy metals not only by synthesizing PCs, but also by contributing to callose deposition. In line with the contribution of callose in counteracting Cd toxicity, we found that another callose-defective mutant, , was more sensitive to high concentrations of Cd than wild-type plants. Moreover,  plants were more susceptible than wild-type to the hemibiotrophic bacterial pathogen . The metabolome also revealed differences in the relative levels of hydroxycinnamic acids and flavonols, with consequences on cell wall properties and auxin content, respectively. First, increased lignification in the  stems was found, probably aimed at counteracting the entry of Cd into the inner tissues. Second, in  shoots, increased relative levels of kaempferol 3,7 dirhamnoside and quercetin hexoside rhamnoside were detected. These flavonols are endogenous inhibitors of auxin transport ; auxin levels in both roots and shoots of the  mutant were in fact lower than those of the wild-type. Overall, our data highlight novel aspects of AtPCS1 functions in .

The  Knockout Mutant for  () Is Defective in Callose Deposition, Bacterial Pathogen Defense and Auxin Content, But Shows an Increased Stem Lignification.

The virulence and broad host range of Fusarium graminearum is associated with its ability to secrete an arsenal of phytotoxic secondary metabolites, including the regulated mycotoxins belonging to the deoxynivalenol family. The TRI genes responsible for the biosynthesis of deoxynivalenol and related compounds are usually expressed during fungal infection. However, the F. graminearum genome harbors an array of unexplored biosynthetic gene clusters that are also co-induced with the TRI genes, including the nonribosomal peptide synthetase 8 ( NRPS8) gene cluster. Here, we identify two bicyclic lipopeptides, gramillin A (1) and B (2), as the biosynthetic end products of NRPS8. Structural elucidation by high-resolution LC-MS and NMR, including H-N-C HNCO and HNCA on isotopically enriched compounds, revealed that the gramillins possess a fused bicyclic structure with ring closure of the main peptide macrocycle occurring via an anhydride bond. Through targeted gene disruption, we characterized the GRA1 biosynthetic gene and its transcription factor GRA2 in the NRPS8 gene cluster. Further, we show that the gramillins are produced in planta on maize silks, promoting fungal virulence on maize but have no discernible effect on wheat head infection. Leaf infiltration of the gramillins induces cell death in maize, but not in wheat. Our results show that F. graminearum deploys the gramillins as a virulence agent in maize, but not in wheat, thus displaying host-specific adaptation.

Gramillin A and B: Cyclic Lipopeptides Identified as the Nonribosomal Biosynthetic Products of Fusarium graminearum.

Fusarium graminearum responds to environmental cues to modulate its growth and metabolism during wheat pathogenesis. Nitrogen limitation activates virulence-associated behaviours in F. graminearum including mycotoxin production and penetrative growth. In other filamentous fungi, nitrogen sensing is mediated by both the Target of Rapamycin (TOR) and the glutamine synthetase (GS)-dependent signaling pathways. While TOR-dependent nitrogen responses have been demonstrated in F. graminearum, the involvement of GS remains unclear. Our study indicates that both the TOR and GS signalling pathways are involved in nitrogen sensing in F. graminearum and contribute to glutamine-induced mycelial growth. However, neither pathway is required for glutamine-induced repression of the mycotoxin deoxynivalenol (DON) indicating that an additional nitrogen sensing pathway must exist. Further, two genes FgBMH1 and FgBMH2 encoding 14-3-3 proteins regulate nitrogen responses with effects on gene expression, DON production and mycelial growth. Unlike yeast, where 14-3-3s function redundantly in regulating nitrogen sensing, the 14-3-3 proteins have differing functions in F. graminearum. While both FgBMH1 and FgBMH2 regulate early glutamine-induced DON repression, only FgBMH2 is involved in regulating reproduction, virulence and glutamine-induced AreA repression. Together, our findings help to clarify the nitrogen sensing pathways in F. graminearum and highlight the involvement of 14-3-3s in the nitrogen response of filamentous fungi.

Two 14-3-3 proteins contribute to nitrogen sensing through the TOR and glutamine synthetase-dependent pathways in Fusarium graminearum.

Deoxynivalenol (DON) is a mycotoxin virulence factor that promotes growth of the  fungus in wheat floral tissues. To further our understanding of the effects of DON exposure on plant cell function, we characterized DON-induced transcriptional changes in wheat spikelets. Four hundred wheat genes were differentially expressed during infection with wild-type  as compared with a  mutant strain that is unable to produce DON. Most of these genes were more induced by the DON-producing strain and included genes involved in secondary metabolism, signaling, transport, and stress responses. DON induction was confirmed for a subset of the genes, including , by treating tissues with DON directly. Previous work indicates that the  ortholog represses trichothecene-induced defense responses and bacterial resistance in , but the role of the NFXL family has not been studied in wheat. We observed greater DON-induced  gene expression in a susceptible wheat genotype relative to the -resistant genotype Wuhan 1. Functional testing using both virus-induced gene silencing and CRISPR-mediated genome editing indicated that  represses  resistance. Together, this suggests that targeting the  gene may help to develop disease resistance in cultivated wheat.

Genome Editing of a Deoxynivalenol-Induced Transcription Factor Confers Resistance to  in Wheat.

Fungal pathogens survive harsh environments and overcome physical, temporal, and chemical barriers to colonize their hosts and reproduce.  was one of the first fungal plant pathogens for which transcriptomic tools were developed, making analysis of gene expression a cornerstone approach in studying its biology. The analysis of gene expression in diverse in vitro conditions and during infection of different cereal crops has revealed subsets of both unique and shared transcriptionally regulated genes. Together with genetic studies, these approaches have enhanced our understanding of the development and infection cycle of this economically important pathogen. Here, we will outline recent advances in transcriptional profiling during sporogenesis, spore germination, vegetative growth, and host infection. Several transcriptional regulators have been identified as essential components in these responses and the role of select transcription factors will be highlighted. Finally, we describe some of the gaps in our understanding of  biology and how expression analysis could help to address these gaps.

Regulation and Dynamics of Gene Expression During the Life Cycle of .

Plants perceive externally produced microbe-associated molecular patterns (MAMPs) and endogenously produced danger-associated molecular patterns (DAMPs) to activate inducible immunity. While several inducible immune responses have been observed during Fusarium graminearum infection, the identity of the signaling pathways involved is only partly known. We screened 227 receptor kinase and innate immune response genes in Arabidopsis to identify nine genes with a role in F. graminearum resistance. Resistance-promoting genes included the chitin receptors LYK5 and CERK1, and the reactive oxygen species (ROS)-producing NADPH oxidase RbohF, which were required for full inducible immune responses during infection. Two of the genes identified in our screen, APEX and the PAMP-induced peptide 1 (PIP1) DAMP receptor RLK7, repressed F. graminearum resistance. Both RbohF and RLK7 were required for full chitin-induced immune responses and PIP1 precursor expression was induced by chitin and F. graminearum infection. Together, this indicates that F. graminearum resistance is mediated by MAMP and DAMP signaling pathways and that chitin-induced signaling is enhanced by PIP1 perception and ROS production.

MAMP and DAMP signaling contributes resistance to Fusarium graminearum in Arabidopsis.

Arbuscular mycorrhizal fungi (AMF) are prominent root symbionts that can carry thousands of nuclei deriving from two parental strains in a large syncytium. These co-existing genomes can also vary in abundance with changing environmental conditions. Here we assemble the nuclear genomes of all four publicly available AMF heterokaryons using PacBio high-fidelity and Hi-C sequencing. We find that the two co-existing genomes of these strains are phylogenetically related but differ in structure, content and epigenetics. We confirm that AMF heterokaryon genomes vary in relative abundance across conditions and show this can lead to nucleus-specific differences in expression during interactions with plants. Population analyses also reveal signatures of genetic exchange indicative of past events of sexual reproduction in these strains. This work uncovers the origin and contribution of two nuclear genomes in AMF heterokaryons and opens avenues for the improvement and environmental application of these strains.

Arbuscular mycorrhizal fungi heterokaryons have two nuclear populations with distinct roles in host-plant interactions.

Author Correction: Arbuscular mycorrhizal fungi heterokaryons have two nuclear populations with distinct roles in host-plant interactions.

Microbial plant pathogens deploy amphipathic cyclic lipopeptides to reduce surface tension in their environment. While plants can detect these molecules to activate cellular stress responses, the role of these lipopeptides or associated host responses in pathogenesis are not fully clear. The gramillin cyclic lipopeptide is produced by the Fusarium graminearum fungus and is a virulence factor and toxin in maize. Here, we show that gramillin promotes virulence and necrosis in both monocots and dicots by disrupting ion balance across membranes. Gramillin is a cation-conducting ionophore and causes plasma membrane depolarization. This disruption triggers cellular signaling, including a burst of reactive oxygen species (ROS), transcriptional reprogramming, and callose production. Gramillin-induced ROS depends on expression of host ILK1 and RBOHD genes, which promote fungal induction of virulence genes during infection and host susceptibility. We conclude that gramillin's ionophore activity targets plant membranes to coordinate attack by the F. graminearum fungus.

A cyclic lipopeptide from Fusarium graminearum targets plant membranes to promote virulence.

Barley is the third most important cereal crop in terms of production in Canada, and Fusarium head blight (FHB) is one of the main fungal diseases of barley. FHB is caused by a species complex of Fusaria, of which  Schwabe is the main causal species of FHB epidemics in Canada. Field surveys show that two or more  species often co-exist within the same field or grain sample, and  is reported as another important species in barley. This study aimed to determine the pathogenicity of , , and a co-inoculation of both species causing FHB in barley. Two susceptible barley cultivars were spray-inoculated at 10 to 14 days after heading. Phenotypic disease severity was rated on a scale of 0-9 at 4, 7, 14, 21, and 28 days after inoculation. There was a significant difference in FHB severity between  and , where infection with  produced more severe disease ratings.  generated lower disease ratings and was not statistically different from the control. When heads were co-inoculated with both  species, the resulting FHB severity was unchanged relative to heads inoculated with  only. The ratio of  to  genomic DNA was also no different than when heads were inoculated with  alone, as quantified with ddPCR using markers specific to each species. The metabolomic analysis of sample extracts showed that -associated metabolites dominated the mycotoxin profile of co-inoculated samples, which corroborated our other findings where  appeared to outcompete  in barley. No significant effect on visual FHB disease ratings or fungal DNA detection was observed between the cultivars tested. However, there were some metabolome differences between cultivars in response to the challenge by both  and .

Pathogenicity and Metabolomic Characterization of  and  Challenge in Barley under Controlled Conditions.

Diverse plant species detect bacterial flagellin using leucine-rich repeat receptor-like kinases to activate defense responses and promote resistance against bacterial invasion. In barley, the impact of flagellin on inducible defense responses has not been fully described. Here, we report that the flagellin-derived flg22 peptide induces callose, reactive oxygen species (ROS) bursts, and differential expression of 2,603 genes in barley. Cultivated barley genotypes produce different amplitudes of flg22-induced ROS bursts, indicating genetic variation for microbe-associated molecular pattern responses. Association mapping revealed a region on chromosome 7 underlying variation in flg22-induced ROS bursts, which contains 12 flg22-induced genes. Genotypes with higher flg22 responses had higher induced resistance to  pv.  (), the causal organism of bacterial leaf streak. The flg22 region of  flagellin evades detection in both barley and Arabidopsis, and a single amino acid change can restore immunogenicity of the peptide. Together, this work underlines the effectiveness of the flagellin-triggered defenses in promoting barley resistance to , which evades recognition of the flg22 region in its host. [Formula: see text]Copyright © 2025 His Majesty the King in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada. This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Transcriptional Profiling and Genetic Mapping of Barley Responses to Bacterial Flagellin.

Seed dormancy is regulated by a combination of developmental and environmental cues to ensure seedling survival in a changing environment. In barley (Hordeum vulgare L.), the SD1 and SD2 (where SD is standard deviation) loci regulate dormancy and pre-harvest sprouting (PHS), though their role in physiological development remains unclear. Malting barley production in Eastern Canada is currently limited due to the high potential for PHS in the region. To understand what genetic loci might be influencing dormancy in Eastern Canadian barley, we evaluated the LegCi biparental population, which was derived from the Léger variety. A quantitative trait loci close to the SD1 on chromosome 5 locus was identified as regulating germination in LegCi, suggesting that the alanine aminotransferase gene (AlaAT1), which underlies dormancy regulation at SD1, influences dormancy in LegCi. Alanine aminotransferases influence energy production in the cell, particularly during nitrogen limitation or oxygen deprivation. LegCi genotypes segregating for dormancy at the SD1 allele showed no differences in abscisic acid or GA-dependent gene expression during grain fill but varied for hypoxia-induced gene expression. Hypoxia suppressed germination in all genotypes but had a significantly higher impact on genotypes with the dormant AlaAT1 relative to genotypes with the non-dormant AlaAT1. This trend was not dependent on the presence of the hull, suggesting that signaling or metabolism inside the germinating seed is influencing hypoxia sensitivity. This work suggests that the non-dormant allele of SD1 is associated with reduced hypoxia stress sensitivity to promote germination. Further work is needed to determine if this trend extends to other barley genotypes.

Article	Year
Insights into the Structure, Function, and Ion-Mediated Signaling Pathways Transduced by Plant Integrin-Linked Kinases. Frontiers in plant science\| PubMed ID: 28421082	2017
The Knockout Mutant for () Is Defective in Callose Deposition, Bacterial Pathogen Defense and Auxin Content, But Shows an Increased Stem Lignification. Frontiers in plant science\| PubMed ID: 29403524	2018
Gramillin A and B: Cyclic Lipopeptides Identified as the Nonribosomal Biosynthetic Products of Fusarium graminearum. Journal of the American Chemical Society\| PubMed ID: 30395461	2018
Two 14-3-3 proteins contribute to nitrogen sensing through the TOR and glutamine synthetase-dependent pathways in Fusarium graminearum. Fungal genetics and biology : FG & B\| PubMed ID: 31605748	2020
Genome Editing of a Deoxynivalenol-Induced Transcription Factor Confers Resistance to in Wheat. Molecular plant-microbe interactions : MPMI\| PubMed ID: 31790345	2020
Regulation and Dynamics of Gene Expression During the Life Cycle of . Phytopathology\| PubMed ID: 32460691	2020
MAMP and DAMP signaling contributes resistance to Fusarium graminearum in Arabidopsis. Journal of experimental botany\| PubMed ID: 34405877	2021
Arbuscular mycorrhizal fungi heterokaryons have two nuclear populations with distinct roles in host-plant interactions. Nature microbiology\| PubMed ID: 37884816	2023
Author Correction: Arbuscular mycorrhizal fungi heterokaryons have two nuclear populations with distinct roles in host-plant interactions. Nature microbiology\| PubMed ID: 37989883	2024
A cyclic lipopeptide from Fusarium graminearum targets plant membranes to promote virulence. Cell reports\| PubMed ID: 38970790	2024
Pathogenicity and Metabolomic Characterization of and Challenge in Barley under Controlled Conditions. Journal of fungi (Basel, Switzerland)\| PubMed ID: 39452622	2024
Transcriptional Profiling and Genetic Mapping of Barley Responses to Bacterial Flagellin. Molecular plant-microbe interactions : MPMI\| PubMed ID: 40062987	2025
Alanine aminotransferase contributes to hypoxia sensitivity and dormancy in barley seeds. The plant genome\| PubMed ID: 40538307	2025

Elizabeth K. Brauer

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